Mechanistic Study of the Inhibitory Effects of Hedyotis diffusa Ethanol Extract on Breast Cancer Cells via Activation of the Cyt-c/Caspase-9–Related Mitochondrial Pathway
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摘要:
目的 探究白花蛇舌草醇提取物（hedyotis diffusa extract，HDE）通过细胞色素 c（cytochrome c，Cyt-c）/半胱天冬酶-9（cysteine aspartate aminotransferase-9，Caspase-9）通路对乳腺癌细胞增殖、迁移及线粒体损伤的影响。 方法 细胞计数试剂盒-8（cell counting Kit-8，CCK-8）检测各组乳腺癌细胞活性。继续培养乳腺癌细胞MCF7，分为对照组、HDE组及过表达Cyt-c组。免疫荧光染色检测MCF7细胞中Cyt-c、Caspase-9蛋白水平。5-乙炔基-2'-脱氧尿苷（5-ethynyl-2´-deoxyuridine，EdU）试剂盒检测MCF7细胞增殖能力。划痕实验检测MCF7细胞迁移能力。流式细胞术检测MCF7细胞线粒体膜电位水平。酶联免疫吸附（enzyme-linked immunosorbent assay，ELISA）试剂盒检测MCF7细胞三磷酸腺苷（adenosine triphosphate，ATP）含量。 结果 与对照组相比，HDE组乳腺癌细胞BT474、SKBr-3、ZR-75-30、MCF7、MDA-MB-453细胞活性降低（P < 0.05）。选择乳腺癌细胞MCF7进行后续研究。与对照组相比，HDE组乳腺癌细胞MCF7细胞Cyt-c、Caspase-9表达增加（P < 0.05），细胞增殖及迁移能力下降（P < 0.05），线粒体膜电位降低，ATP含量减少（P < 0.05）。与对照组相比，过表达Cyt-c组乳腺癌细胞MCF7细胞增殖及迁移能力下降（P < 0.05），线粒体膜电位降低，ATP含量减少（P < 0.05）。 结论 HDE可能通过激活Cyt-c/Caspase-9通路抑制乳腺癌细胞增殖、迁移，并促进线粒体损伤。 -
关键词:
- 乳腺癌 /
- 白花蛇舌草醇提取物 /
- Cyt-c/Caspase-9通路 /
- 线粒体损伤
Abstract:Objective The effects of Hedyotis diffusa extract (HDE) on proliferation, migration and mitochondrial damage of breast cancer cells were investigated by using cytochrome c（Cyt-c）/cysteine aspartate aminotransferase-9（Caspase-9）pathway. Methods Cell Counting Kit-8 （CCK-8） was used to detect breast cancer cell viability in each group. MCF7 breast cancer cells were continuously cultured and divided into control group, HDE group, and Cyt-c overexpression group. Immunofluorescence staining was used to detect the protein levels of Cyt-c and Caspase-9 in MCF7 cells. 5-ethynyl-2'-deoxyuridine （EdU） kit was used to detect MCF7 cell proliferation ability. Scratch assay was used to detect MCF7 cell migration ability. Flow cytometry was used to detect mitochondrial membrane potential levels in MCF7 cells. Enzyme-linked immunosorbent assay （ELISA） kit was used to detect adenosine triphosphate （ATP） content in MCF7 cells. Results Compared with the control group, the cell viability of BT474, SKBr-3, ZR-75-30, MCF7 and MDA-MB-453 cells in the HDE group was decreased （P < 0.05）. MCF7 breast cancer cells were selected for subsequent studies. Compared with the control group, the HDE group showed increased expression of Cyt-c and Caspase-9 in MCF7 cells （P < 0.05）, decreased cell proliferation and migration ability （P < 0.05）, reduced mitochondrial membrane potential, and decreased ATP content （P < 0.05）. Compared with the control group, the Cyt-c overexpression group showed decreased cell proliferation and migration ability （P < 0.05）, reduced mitochondrial membrane potential, and decreased ATP content （P < 0.05）. Conclusion HDE may inhibit breast cancer cell, and promote mitochondrial damage by activating Cyt-c/Caspase-9 pathway. -
Key words:
- Breast cancer /
- Hedyotis diffusa extract /
- Cyt-c/Caspase-9 pathway /
- Mitochondrial damage
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图 2 各组乳腺癌细胞BT474、SKBr-3、ZR-75-30、MCF7、MDA-MB-453的细胞活性（$\bar x \pm s $，n = 3）
A：BT474细胞活性比较；B：SKBr-3细胞活性比较；C：ZR-75-30细胞活性比较；D：MCF7细胞活性比较；E：MDA-MB-453细胞活性比较；与对照组比较，*P < 0.05。
Figure 2. Cell viability of breast cancer cell lines BT474，SKBr-3，ZR-75-30，MCF7，and MDA-MB-453 in each group（$\bar x \pm s $，n = 3）
图 3 乳腺癌细胞MCF7中Cyt-c、Caspase-9蛋白水平（$\bar x \pm s $，n = 3）
A：乳腺癌细胞MCF7中Cyt-c、Caspase-9、Cleaved-caspase3蛋白印迹图；B：乳腺癌细胞MCF7中Cyt-c蛋白定量图；C：乳腺癌细胞MCF7中Caspase-9蛋白定量图；D：乳腺癌细胞MCF7中Cleaved-caspase3蛋白定量图；与对照组比较，***P < 0.001；与EV组比较，###P < 0.001。
Figure 3. Protein levels of Cyt-c and Caspase-9 in MCF7 breast cancer cells （$\bar x \pm s $，n = 3）
图 4 乳腺癌细胞增殖能力、迁移能力、线粒体膜电位及ATP含量（$\bar x \pm s $，n = 3）
A-B：EdU试剂盒检测乳腺癌细胞增殖能力；C-D：划痕实验检测细胞迁移能力及相对迁移距离比较；E：流式细胞术检测线粒体膜电位；F-G：流式细胞术检测细胞凋亡率；H：ELISA试剂盒检测各组细胞ATP含量；与对照组比较，***P < 0.001；与EV组比较，###P < 0.001。
Figure 4. Breast cancer cell proliferation capacity，migration capacity，mitochondrial membrane potential，and ATP content（$\bar x \pm s $，n = 3）
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